Short multi-repeat rna targeting gene silencing

Abstract

Short multi-repeat RNA targeting constructs for manipulating RNA targets and obtaining variable levels of gene silencing, and methods of using the same to silence RNA targets in mammals, insects, plants, and fungus. The constructs comprise two or more distinct guide nucleotide sequences (in the absence of CRISPR nuclease) that are complementary to one or more RNA targets, wherein each guide nucleotide sequence consists of a single-stranded antisense nucleotide fragment of 100 nt or less.

1 . Short multi-repeat RNA targeting constructs for manipulating one or more RNA targets, said constructs comprising two or more distinct guide nucleotide sequences that are complementary to one or more RNA targets, wherein each guide nucleotide sequence consists of a single-stranded antisense nucleotide fragment of 100 nt or less. 
     
     
         2 . The short multi-repeat RNA targeting constructs of  claim 1 , wherein each guide nucleotide sequence consists of a single-stranded antisense nucleotide fragment of 15 nt to 100 nt. 
     
     
         3 . The short multi-repeat RNA targeting constructs of  claim 1 , wherein each guide nucleotide sequence consists of a single-stranded antisense nucleotide fragment of 80 nt or less. 
     
     
         4 . The short multi-repeat RNA targeting constructs of  claim 1 , wherein each guide nucleotide sequence consists of a single-stranded antisense nucleotide fragment of 50 nt or less. 
     
     
         5 . The short multi-repeat RNA targeting constructs of  claim 1 , wherein each guide nucleotide sequence consists of a single-stranded antisense nucleotide fragment of 22 nt to 30 nt. 
     
     
         6 . The short multi-repeat RNA targeting constructs of  claim 1 , wherein said guide nucleotide sequences are guide RNA (crRNA) or DNA encoding for the guide RNA, which are capable of binding or hybridizing to the RNA target. 
     
     
         7 . The short multi-repeat RNA targeting constructs of  claim 1 , wherein the RNA target is a coding sequence, preferably mRNA or pre-mRNA, or a non-coding sequence, preferably ncRNA, IncRNA, tRNA, or rRNA. 
     
     
         8 . The short multi-repeat RNA targeting constructs of  claim 1 , wherein said guide nucleotide sequences each bind to a different region of the same RNA target. 
     
     
         9 .- 10 . (canceled) 
     
     
         11 . The short multi-repeat RNA targeting constructs of  claim 1 , wherein said guide nucleotide sequences each bind to a different RNA target. 
     
     
         12 . (canceled) 
     
     
         13 . The short multi-repeat RNA targeting constructs of  claim 1 , wherein said constructs are comprised within a single expression cassette, wherein each of said two or more distinct guide nucleotide sequences are:
 directly linked on said expression cassette; or   indirectly linked, such that each guide nucleotide sequence is separated by an intervening sequence on said expression cassette.   
     
     
         14 .- 16 . (canceled) 
     
     
         17 . The short multi-repeat RNA targeting constructs of  claim 13 , wherein said single expression cassette is a homogenous multimer wherein each of said two or more distinct guide nucleotide sequences consists of the same sequence and has complementarity with the same region of the same RNA target or complementarity with a homologous region of a different RNA target. 
     
     
         18 . The short multi-repeat RNA targeting constructs of  claim 17 , wherein said homogenous multimer comprises up to eight repeats of the same guide nucleotide sequence. 
     
     
         19 . The short multi-repeat RNA targeting constructs of  claim 13 , wherein said single expression cassette is a heterogenous multimer wherein each of said two or more distinct guide nucleotide sequences consists of different sequences having complementarity with different RNA targets or different regions of the same RNA target. 
     
     
         20 . The short multi-repeat RNA targeting constructs of  claim 13 , wherein said constructs are located on the expression cassette adjacent at least one regulatory element. 
     
     
         21 . The short multi-repeat RNA targeting constructs of  claim 1 , wherein said constructs are comprised on respective separate expression cassettes, wherein said constructs are configured for co-expression. 
     
     
         22 . The short multi-repeat RNA targeting constructs of  claim 1 , said constructs being free of nuclease-encoding nucleotide sequences, or nuclease protein. 
     
     
         23 . (canceled) 
     
     
         24 . The short multi-repeat RNA targeting constructs of  claim 1 , said constructs comprising three or more of said distinct guide nucleotide sequences. 
     
     
         25 . A composition for manipulation of RNA targets, said composition comprising a plurality of short multi-repeat RNA targeting constructs according to  claim 1  dispersed in a carrier or vehicle. 
     
     
         26 . A method of modifying an RNA target, said method comprising delivering to said RNA target a short multi-repeat RNA targeting construct according to  claim 1 . 
     
     
         27 .- 37 . (canceled) 
     
     
         38 . A method of gene silencing comprising delivering to a cell, tissue, organ, or organism, a short multi-repeat RNA targeting construct according to  claim 1 , wherein at least one of said two or more distinct guide nucleotide sequences is complementary to an RNA target associated with said gene. 
     
     
         39 . A method of manipulating a plant characteristic, said method comprising delivering to a plant cell, tissue, or plant a short multi-repeat RNA targeting construct according to  claim 1 , wherein at least one of said two or more distinct guide nucleotide sequences is complementary to an RNA target associated with a gene encoding for said plant characteristic. 
     
     
         40 .- 42 . (canceled) 
     
     
         43 . A seed, seedling, or progeny of a plant produced according to the method of  claim 39 , wherein said seed, seedling, or progeny expresses said short multi-repeat RNA targeting construct. 
     
     
         44 .- 45 . (canceled)